The proposed studies will evaluate the impaired endothelial cell dependent dilation and enhanced vasoconstrictor responses which occur in the diabetic microvasculature. The arteriolar response to acetylcholine, mediated by endothelial derived relaxing factor(EDRF), is greatly suppressed. This could be due to inadequate production or inactivation of EDRF as well as competition by constrictor stimuli. The suppression of EDRF dilation occurs in about one week in streptozotocin diabetic rats and is duplicated in normal arterioles by one hour of local exposure to isotonic hyperglycemia (300-500 mg%). Pretreatment with superoxide dismutase protects EDRF function during exposure to 500 mg% glucose, and post-treatment substantially restores EDRF function. These results suggest that oxygen or hydroxyl radicals produced in response to hyperglycemia may be the mechanism(s) responsible for EDRF suppression. Specific scavengers of oxygen or hydoxyl radicals will be used to determine which radical species is primarily responsible for impaired EDRF function in acute and chronic hyperglycemia. A potential source of radicals is increased ecosanoid synthesis during hyperglycemia; cycloxygenase, inhibition partially restores EDRF function in diabetic rats. If cycloxygenase inhibition substantially decreases radical formation during hyperglycemia, eicosanoid synthesis will be implicated as the primary source of radicals. The extent to which loss of EDRF function, hyperglycemia, and decreased insulin action contribute to the enhanced vasoconstrictor responses in diabetic rats is not known. The potential contributions of each factor to the constrictor responses to norepinephrine, angiotensin II, and myogenic pressor stimuli, all of which act directly on vascular smooth muscle cells, will be determined. EDRF function of normal arterioles will be suppressed with an arginine analog(NMMA) or local isotonic hyperglycemia so that the modulation of constrictor regulation by EDRF and additional complications caused by hyperglycemia can be determined. Comparison of EDRF and constrictor functions in Zucker diabetic rats maintained in a hyperglycemic insulin-resistant or normoglycemic insulin-resistant state will be used to determine whether hyperglycemia or insulin-resistance primarily influences altered vascular regulation. In vitro studies indicate that hyperglycemia increases sorbitol formation by 2 to 3-fold in endothelial cells. Conversion of excess glucose to sorbitol by aldose reductase may be an attempt to protect the intracellular environment from hyperglycemia. Whether aldose reductase expression is increased in microvascular cells during diabetic hyperglycemia is not known. Vascular cells may initially increase expression of aldose reductase to compensate for the excess glucose, but eventually, cell damage may attenuate expression, leading to unchecked cytoplasmic hyperglycemia. Expression of aldose reductase mRNA in intestinal microvessels of early and advanced insulin-resistant and dependent diabetic rats will be measured using in situ hybridization. These results will be used to determine the time-dependent expression of aldose reductase, temporal correlation to disturbances in microvascular regulation, and whether the cause of hyperglycemia influences aldose reductase expression.